An investigation into an outbreak of pancytopenia in cats in the United Kingdom

Abstract Background In spring 2021 increasing numbers of cats presenting with severe pancytopenia were noted in United Kingdom (UK). Objective To describe process and outcome of the investigation performed into the outbreak of pancytopenia in cats. Animals Five hundred and eighty client owned cats that presented with severe bi‐ or pancytopenia of unknown cause. Methods Real‐time data collection was performed by an online registration forum available to all veterinary surgeons in UK. Data collected included demographics, clinicopathological findings, diagnostic testing, dietary and drug history, outcome and COVID household status. Mycotoxicological feed analysis was performed on feed samples of 3 diets frequently mentioned in the database and 3 control diets. Results Five hundred and eighty cats presented to 378 veterinary practices were included for analysis. Case fatality rate was 63.3%. Dietary history was available for 544 (93.8%) cats, of which 500 (86%) were fed 1 of 3 diets (which were recalled midinvestigation). 54 (9.3%) cats were not fed a recalled product, with diet information unknown in 26 (4.5%) cats. Analysis of feed samples revealed concentrations of hematotoxic trichothecene T‐2/HT‐2 mycotoxins greater than recommended by the European Commission in 5/7 recalled diet samples but in none of control diet samples. The trichothecene mycotoxin diacetoxyscirpenol (DAS) was detectable in all recalled diet samples but not in any of control samples. Conclusion and Clinical Importance Contaminated‐feed induced trichothecene mycotoxicosis should be considered as a differential diagnosis for pancytopenia in cats.

destruction or consumption of blood cells, or a combination of these, or by a failure of the bone marrow to produce. 1 Causes of pancytopenia in cats include infectious diseases (eg, leukemia, immunodeficiency, and panleukopenia virus infection), neoplasia (eg, lymphoma, leukemia, hemophagocytic syndrome), toxicosis (eg, drugs, radiation), deficiencies in essential minerals and vitamins (eg, cobalamin deficiency), immune-mediated, and idiopathic disease. 2 including cats that shared households, presented to the authors' hospital, with a few days' history of lethargy, anorexia, and bleeding tendencies. All cats had severe bi-or pancytopenia; and an underlying cause could not be identified despite thorough investigations. All 7 cats died or were euthanized because of ongoing hemorrhage.
Additionally, advice enquiries from first opinion as well as other referral hospitals in regard to similar presentations were received at an unusually high frequency.
In the UK, clear guidance for the management of communicable disease outbreaks and for foodborne illness exist for illnesses affecting people. 7,8 However, the management of outbreaks of veterinary diseases has not been described in small animal practice. This report aims to describe both the investigation of the outbreak and its findings, and the affected cohort of cats. Letters explaining the nature of the investigation and containing the link to the questionnaire were also sent to veterinary trade journals. 9 Data collected included date of presentation, signalment, duration and type of clinical signs, indoor/outdoor status, clinical pathological findings, bone marrow sampling details, feline immunodeficiency (FIV), feline leukemia (FeLV) and feline panleukopenia (FPLV) virus testing results, parasite control medication, details of any other recent medication or vaccination, cat litter type used, and dietary information for each cat affected. Details on owner household location (through the first part of the owner postcode), any history of COVID 19 infection in the household and whether any other household pets were affected were also gathered. Ethical approval for the study was granted by the institutional ethics and welfare committee (URN SR2021-0148).

| Criteria for case inclusion
Data from the questionnaire was compiled into a database and cases individually examined. Cats were considered to be eligible for inclusion if they presented with a leukopenia (<5.5 Â 10 9 /L; or neutrophil concentration <2.5 Â 10 9 /L) or thrombocytopenia (<150 Â 10 9 /L), or a combination of these, with or without an anemia (hematocrit or packed cell volume <27%). Cats that presented with anemia without concurrent thrombocytopenia were excluded. If data on complete blood cell (CBC) analysis was not available, the response was excluded. Responses were included even if other sections were incomplete. Responses were excluded in the final analysis if they were received from countries other than the UK. Registered cats were excluded if follow-up information provided by their primary care practice revealed an underlying disease process that could explain changes in blood cell concentrations (eg, neoplasia).

| Feed sample analysis
Packages of pelleted dry feed of specific batch numbers of feeds that were frequently mentioned in the database and packages of commercial dry diets ("control samples"), which appeared infrequently in the database, were analyzed for trichothecene mycotoxin content includ-

| Statistical analysis
Following data checking for internal validity, identifying and correcting duplicate, incorrectly formatted and irrelevant data in Excel (Microsoft Office Excel 2013, Microsoft Corp), analyses were conducted using SPSS version 28.0 (IBM Corp). All continuous variables were nonnormally distributed and so were summarized using median and range. When data was compared between different groups Mann-Whitney U test, chi-square test and Fisher's exact test were used as appropriate.
Owner partial postcodes were geocoded using a free web-based resource. 10

| Geographical distribution of affected cats
Seven partial logged postcodes were incorrect and therefore excluded from case mapping. Owners of affected cats lived throughout the UK, although the majority were based in the Midlands, South-East England, and London ( Figure 2).

| Diet
In total, 44 diet brands were fed to the 554 cats with brand information recorded, with 3 diet brands (brands A, B, and C) predominating ( Figure 3). These 3 brands were recalled from the market on 16 June 2021. 12 There were 500 (86.2%) cats fed a recalled product, while 54 (9.3%) cats were not fed a recalled product, with diet information unknown in 26 (4.5%) cats. Of the cats that were not fed a recalled product, 38 (70.4%) had indoor and outdoor access, while 16 (29.6%) were indoor only. There had been a recent diet change in 106 (18.3%) cats, no recent change in 472 (81.4%) cats, while this information was not recorded in 2 (.34%) cats.
Of the cats that were not fed a recalled product, a recent diet change was reported for 11, including 4 of the 16 indoor cats.  Figure 4). On or before food recall, 2 cats (0.6%) were presented by the owner as they were fed a recalled diet, while after food recall 37 cats (15.4%) were presented by the owner for the same reason. In addition, 8 cats (2.4%) presenting on or before food recall were presented as a housemate was diagnosed with pancytopenia, while 15 cats (6.3%) presenting after food recall were presented as a housemate was diagnosed with pancytopenia.
The duration of clinical signs, total white blood cell concentration, neutrophil concentration, platelet concentration, PCV and case fatality were compared between cats presenting on or before the date of food recall and after food recall (Table 1).
Of cats presenting on or before the date of food recall with diet information available, 298/319 (93.4%) were fed a recalled diet, while 21 (6.6%) were not fed a recalled diet. This differed to cats presenting after food recall, whereby a lesser proportion (202/235; 86.0%) of cats were fed a recalled diet, while a greater proportion (33; 14.0%) were not fed a recalled diet.

| Feed analysis
The trichothecene feed analysis revealed T-2/HT-2 sum concentration higher than the recommended guidance value in 5 of 7 analyzed dry feed packages of recalled brands ( Table 2). 13 Two of 3 control samples did not contain any detectable concentration of T-2/HT-2 and 1 of the control samples contained detectable T-2/HT-2 but at a concentration below the recommended guidance values (Table 2). 13 DAS was also detectable in all 7 samples of recalled brands but not in any of the control samples (Table 2).
F I G U R E 3 Most common diet brands (%) fed to affected cats with brand information recorded (n = 554)

| Liver tissue analysis
The nontargeted chemical testing of postmortem liver samples found no single feature present in all livers from 5 affected cats.
There was no evidence of any known toxin or metabolite of any known toxin. Specific targeted metabolite searches found peaks indicative of mycotoxin exposure, specifically T-2 toxin, and DAS.

| DISCUSSION
The clinical pathological findings of the cats affected in this outbreak were most consistent with an aplastic pancytopenia (formerly also called aplastic anemia), which is characterized by cytopenias of erythrocytes, leukocytes and platelets in the peripheral blood combined with a hypo-or acellular bone marrow with the marrow space being replaced by adipose tissue. 14 Aplastic pancytopenia is a rare disorder in cats which can be caused by destruction of bone marrow stem cells, genetic mutations causing inadequate stem cell function or hemopoietic microenvironment disorders. 14 Idiopathic aplastic pancytopenia is described in people but this has not been reported in cats. 14   less than a quarter of the most popular brand. 16,17 Fifty-four cats (9.3%) were recorded to have been eating a nonrecalled brand. It is possible that some of these cats might never have been exposed to the suspected diets as a minority were reported as indoor cats only. It is also possible that some registered cats had an underlying disease that was not identified because of lack of investigations, that some of the cats accessed a recalled brand outside the home and finally that some cats could have had a recent diet change with the veterinarian only noting the brand that was being fed at the time of presentation without further verification whether a diet change had occurred or not. The latter theory is also suspected to be the reason for the significantly lower proportion of cats being fed a recalled diet after the recall had occurred.
The strong epidemiological link to feed-with over 86% of affected cats being exposed to at least 1 of the recalled dietssuggested either a feed deficiency or intoxication. Diagnostic tests performed on individual cats showed no evidence of vitamin deficiency or heavy metal intoxication. 18 The group A tricothecene myco- to T-2 and its main toxic effects include emesis and hematotoxicity. 29,30 As for T-2/HT-2 glucuronide conjugation is 1 of the main metabolization pathway for DAS, lacking in cats. 30 In mice, the LOAEL of DAS administered orally to cause anorexia is 5 times greater than for either T-2 or HT-2. 31 In dogs, NOAELs for DAS are based on laboratory studies for both chronic and acute exposure but are not reported in cats. 32,33 There is no data describing the levels of DAS found in cat food, but estimated exposure levels based on concentrations in cereal grains and their relative proportions in diets suggest 95th percentile diet concentration of 13 μg/kg in dry cat food and this is therefore thought to be a safe level. 30 The levels of DAS detected in the recalled foods tested were far higher than this 95th percentile and it is suspected that it could have caused adverse effects.
T-2 and HT-2 are considered to be cumulative when assessing risk of toxicosis in feed. 17 Also, dietary co-exposure to multiple toxins produced by Fusarium spp. is thought to be common, 30 with the effects of T-2 and DAS being additive for oral lesions and synergistic in decreasing egg production in laying hens. 34   Although there is a strong association between the ingestion of previous batches of brands A, B, and C and the occurrence of pancytopenia, diet cannot be definitively stated to be the cause as there is no LOAEL established for T-2, HT-2, and DAS in cats. No specific source of mycotoxin was established, although it is recognized that many possible feed ingredients could be responsible in cat food manufacture, particularly wheat, oat and maize; but also root vegetables. 49 Given the high proportion of affected cats known to be consuming the 3 brands, their common manufacturing source, the detection of T-2 and HT-2 in the feeds at levels much higher than those previously reported in cat feeds (and above that recommended by the European Union), the consistency between the clinical signs seen with T-2/HT-2 intoxication in various species including cats and those seen in our cohort, the decreased severity of clinical signs (likely because of decreased toxin exposure) and eventual resolution of the outbreak when the 3 brands were withdrawn from the market, it is reasonable to propose that trichothecene contamination of the recalled food brands was the cause of this pancytopenia outbreak in cats.
In conclusion, trichothecene intoxication should be considered as a possible differential diagnosis for a cat presenting with pancytopenia of unknown cause and if an outbreak were to occur in the future, rapid investigation of feed should be undertaken. This investigation has highlighted the need for introducing standardized testing of cat foods for mycotoxin contamination across the pet food industry and following this the establishment of NOAELs for T2/HT2 and DAS in cats through greater monitoring of pet feed levels. Specialist, and Angelika Rupp, University of Glasgow, for providing a liver tissue sample. We thank our families for their support over this period.